Analysis of leukocyte populations from blood samples is an integral and essential part of diagnostic procedures regarding a multiplicity of pathologies. Measurements of basophils and eosinophils that are subpopulations of leukocytes are important to the diagnosis of several diseases.
Traditional analysis of blood samples involves the smearing of a blood sample on a microscope slide, followed by a visual analysis of the slide. This approach is extremely time consuming as well as being subject to the interpretation of the individual analyzing the slide. These factors have led to the development of automated leukocyte analysis utilizing flow cytometry. An essential step in leukocyte analysis using automated hematology instruments is the lysis of the red blood cells. Thus far, various lytic reagents and automated methods have been developed for use with whole blood samples.
U.S. Pat. No. 4,485,175 (to Ledis et al.) describes a reagent system and method for performing differential leukocyte determinations into three subpopulations utilizing automated cell counting equipment. This reagent system contains a blood diluent and a lytic reagent. The lytic reagent comprises a mixture of quatemary ammonium surfactants. This reagent system differentiates the leukocytes into three subpopulations: lymphocytes, monocytes and granulocytes.
U.S. Pat. No. 5,155,044 (to Ledis et al.) discloses a reagent system and method for the rapid isolation and analysis of leukocytes from a whole blood sample and enables automated differentiation into five subpopulations using an automated hematology analyzer capable of impedance (DC), radio frequency (RF) and light scatter (LS) measurements. The reagent system is composed of an aqueous lytic reagent which comprises an acid, or a mixture of acid and saponin, and an aqueous salt quench solution. This method is rapid and provides a five part leukocyte differential of a whole blood sample in a single step measurement.
U.S. Pat. No. 5,786,224 (to Li et al.) discloses a lytic reagent system and a method for automated differentiation of leukocytes into five subpopulations using impedance, radio frequency and light scatter measurement devices. The lytic reagent system is composed of an aqueous lytic reagent which comprises a polyoxyethylene based nonionic surfactant, SDS and an acid, and a stabilizing reagent which comprises a hypertonic, alkaline reagent composition. The disclosed lytic reagent system differentiates leukocyte into five subpopulations using a single measurement when impedance, radio frequency and light scatter measurements are used.
However, if the above two lytic reagent systems are used on a hematology analyzer equipped with only impedance and light scatter measurement devices, the basophil subpopulations will not be adequately differentiated from lymphocyte subpopulations. If only impedance and radio frequency measurements are used for the blood analysis, eosinophils will not be differentiated from neutrophils due to a complete overlap of the two populations in DC vs. Opacity (a function of DC and RF) scattergram.
Another approach for differentiation of leukocytes into four or five subpopulations is to use multiple reagents and multiple measurements. Separate reagents are used for differentiating the specific subpopulations. Typically, a first lytic reagent, like the ones revealed in U.S. Pat. No. 5,116,539 (to Hamaguchi et al.) and U.S. Pat. No. 5,389,549 (to Hamaguchi et al.) are used to differentiate the leukocytes into three subpopulations (i.e., monocytes, lymphocytes and granulocytes) using impedance and radio frequency measurements.
Hamaguchi et al. (U.S. Pat. No. 5,389,549) discloses a lytic reagent for differentiating leukocytes into the above mentioned three subpopulations using impedance and radio frequency measurements. The lytic reagent consists essentially of a first cytolytic solution having pH of 1.5-5.0 and containing a surfactant in an amount effective to reduce erythrocyte ghosts for distinction from leukocytes without causing undesirable leukocyte damage, wherein the surfactant consists essentially of a polyoxyethylene based nonionic surfactant represented by the formula: EQU R.sub.1 --R.sub.2 --(CH.sub.2 CH.sub.2 O).sub.n --H
wherein R.sub.1 is an alkyl, alkenyl or alkynyl group having 12 to 22 carbon atoms, R.sub.2 is --O--, --OC.sub.6 H.sub.6 -- or --COO--, and n is an integer of 20-100. The reagent further consists essentially of a second cytolytic solution used in combination with the first reagent. The second cytolytic solution has a pH of 5.0-12.0 and an osmolarity of 150-2000 mOsm/kg.
In U.S. Pat. No. 5,116,539, an additional lytic reagent is used with an additional measurement to obtain the eosinophil subpopulation. This enables a four part differentiation of lymphocytes, monocytes, eosinophils and the remaining granulocytes. The lytic reagent is composed of a polyoxyethylene-based nonionic surfactant and a buffer to adjust the pH of the solution within the range of 3-11. The lytic reagent lyses not only red blood cells but also the leukocytes except eosinophils so that the eosinophils can be counted based on their remaining cellular volume measured by DC. However, to achieve eosinophil separations the method of using the lytic reagent requires incubation of the sample mixture at 40.degree. C. for a period of 50 seconds. This elevated temperature requirement necessitated instrumentation which is significantly more complex because the reactions must be thermostatically controlled. In addition, the extended reaction time directly decreases the throughput of the automated analyzer.
In U.S. Pat. No. 5,389,549, two additional lytic reagents are needed and two additional measurements are required to obtain the eosinophil and basophil subpopulations so as to provide a five part differential. More specifically, the additional lytic reagent for differential analysis of basophils is an aqueous solution composed of a polyoxyethylene-based nonionic surfactant, potassium ophthalate, hydrochloric acid and nitric acid. This lytic reagent lyses not only red blood cells but also the leukocytes except basophils so that the basophils can be counted based on their remaining cellular volume measured by DC. Again, to achieve basophil separation the method requires incubation of the sample mixture at elevated temperature. In this disclosure, the total eosinophil and basophil subpopulations obtained from separate measurements are subtracted from the total granulocyte population to obtain the neutrophil subpopulation.
Hamaguchi et al. teach that polyoxyethylene-based nonionic surfactants are moderate in lysing blood and is suitable for leukocyte differential analysis. Hamaguchi et al. further teach that quaternary ammonium salt damage cells violently and it is entirely meaningless to use a quaternary ammonium salt as a cytolytic agent in a method of classifying leukocytes by the combination of the impedance and radio frequency methods.
U.S. Pat. No. 5,196,346 (to Lefevre et al.) discloses a lytic reagent and method of using the same for automated determination of basophils in whole blood samples by lysing all blood cells, including both erythrocytes and leukocytes, with the exception of the basophils. The lytic reagent was composed of a polyoxyethylene ether-type surfactant, a phthalic acid/HCl mixture, sodium dodecyl sulfate (SDS) and a butylated hydroxytoluene-type antioxidizing agent. The disclosed method requires a thermostatically controlled reaction temperature between 30 to 40.degree. C. and is limited to the counting of only basophils by impedance measurement.
U.S. Pat. No. 5,821,128 (to Provost) discloses lytic reagents and method for determination of basophils and eosinophils in whole blood samples. The lytic reagent for basophil measurement consists essentially of polyoxyethylene 9-lauryl ether and acids to maintain pH of the reagent between 2 and 3.5. The method uses the lytic reagent to lyse all blood cells including erythrocytes and leukocytes except basophils. Thereafter, the basophils are counted by impedance measurement. This method requires the lysing reaction to be performed at 40.degree. C. Provost also discloses that by adjusting the pH of the lytic reagent to basic, i.e., between 9 and 10, the lytic reagent enables separation of eosinophils from other cell types by impedance measurement. Two separate reagents and measurements are required to provide basophil and eosinophil differentials.
U.S. Pat. No. 5,518,928 (to Cremins et al.) discloses a method of determination of basophils in whole blood samples by optical measurement. The method uses a non-cationic surfactant and an acid to lyse all blood cells, but retain basophils as intact cells. The lytic reagent contains a C.sub.6 -C.sub.16 aliphatic alcohol ether of a polyoxyethylene glycol and has a pH about 1.8 to about 2.3. The method detects the light scattered by the cells and nucleus after treating a whole blood sample by the lytic reagent and differentiates basophils from other cell types.
Japanese Patent Publication No. Hei 3-20667 (Hamaguchi et al.) discloses a reagent for discriminately detecting leukocytes using impedance measurement. The reagent comprises a polyoxyethylene surfactant represented by the formula: EQU R.sub.1 --R.sub.2 --(CH.sub.2 CH.sub.2 O).sub.n --H
wherein R.sub.1 is an alkyl, alkenyl or alkynyl group having 10 to 25 carbon atoms, R.sub.2 is --O--, --OC.sub.6 H.sub.6 -- or --COO--, and n is an integer of 12-30; and a quaternary ammonium salt represented by the formula: ##STR1##
wherein R.sub.1 is a C.sub.10 -C.sub.20 alkyl group, each of R.sub.2, R.sub.3 and R.sub.4 is C.sub.1 -C.sub.7 alkyl groups and Z.sup.- is halogen atom; and having a pH between 3 and 4. Hamaguchi et al. teach that the sizes of basophils can be maintained at pH 3 or higher by using the reagent which is capable of making other leukocyte subpopulations naked.
U.S. Pat. No. 5,817,518 (to Li et al.) discloses a lytic reagent composition and a method for differentiating eosinophils using impedance and radio frequency measurements. The lytic reagent composition comprises an alkali metal salt of alkyl sulfate, an eosinolytic agent, a nonionic surfactant and a physiological salt. This lytic reagent composition lyses red blood cells and selectively affects and separates the eosinophil subpopulation from the other granulocyte subpopulations by selectively shrinking the eosinophils. Therefore, it enables the differentiation of eosinophils from other cell types when analyzed by impedance and radio frequency measurements. Moreover, Li et al. teaches a lytic reagent system and a method for automated differentiation of leukocytes into five subpopulations using impedance, radio frequency and light scatter measurement devices. The method comprises mixing a first aliquot blood sample with a first lytic reagent to lyse red blood cells and selectively shrink eosinophils, mixing a second aliquot blood sample with a second lytic reagent system to lyse red blood cells and preserve leukocytes, analyzing the treated sample of the first and second aliquots by impedance and radio frequency measurements to differentiate one and four leukocyte subpopulations, respectively; and reporting five leukocyte subpopulations.
U.S. Pat. No. 5,538,893 (to Sakata et al.) discloses a reagent and a method for classifying leukocytes into basophils, immature granulocytes, mononuclear cells and granulocytes other than basophils by measuring the size and a morphological feature of the cells. The reagent comprises at least one nonionic surfactant having an additional mole number of polyoxyethylene of 3 to 9, at least one cationic surfactant, a buffer adjusting a pH value to 2.5 to 4.0, and an alkali metal halide. The cationic surfactant is a pyridinium salt, or a quatemary ammonium salt having a formula: ##STR2##
wherein R.sub.1 is a C.sub.10 -C.sub.18 alkyl or alkenyl group, each of R.sub.2, R.sub.3 and R.sub.4 is C.sub.1 -C.sub.3 alkyl or alkenyl groups and X.sup.- is halogen group. More specifically, the size is measured by forward low angle (1-5.degree.) scattered light or impedance, and morphological features of the cells are measured by light side scattered or the light forward high angle (10-20.degree.) scattered from a cell.
U.S. Pat. No. 5,677,183 (to Takarada et al.) discloses a method for classifying leukocytes into five subpopulations by using two separate lytic reagents. The first reagent enables a differentiation of leukocytes into four subpopulations, and the second reagent enables a differentiation of basophils from other cell types, when the treated sample is analyzed by the size and the morphological features of the cells. Takarada et al. teach that the second reagent is composed of polyoxyethylene (9) dodecyl ether, a quaternary ammonium salt and a buffer to adjust the pH to about 3.3. With this method, the cell size is measured by forward low angle (1-5.degree.) scattered light or impedance, and morphological features of the cells are measured by light side scattered, or the light forward high angle (10-20.degree.) scattered from a cell.